Analysis of substrate specificity of cytochrome P450 monooxygenases involved in trichothecene toxin biosynthesis

Trichothecenes are a structurally diverse family of toxic secondary metabolites produced by certain species of multiple fungal genera. All trichothecene analogs share a core 12,13-epoxytrichothec-9-ene (EPT) structure but differ in presence, absence and types of substituents attached to various positions of EPT. Formation of some of the structural diversity begins early in the biosynthetic pathway such that some producing species have few trichothecene biosynthetic intermediates in common. Cytochrome P450 monooxygenases (P450s) play critical roles in formation of trichothecene structural diversity. Within some species, relaxed substrate specificities of P450s allow individual orthologs of the enzymes to modify multiple trichothecene biosynthetic intermediates. It is not clear, however, whether the relaxed specificity extends to biosynthetic intermediates that are not produced by the species in which the orthologs originate. To address this knowledge gap, we used a mutant complementation-heterologous expression analysis to assess whether orthologs of three trichothecene biosynthetic P450s (TRI11, TRI13 and TRI22) from Fusarium sporotrichioides, Trichoderma arundinaceum, and Paramyrothecium roridum can modify trichothecene biosynthetic intermediates that they do not encounter in the organism in which they originated. The results indicate that TRI13 and TRI22 could not modify the intermediates that they do not normally encounter, whereas TRI11 could modify an intermediate that it does not normally encounter. These findings indicate that substrate promiscuity varies among trichothecene biosynthetic P450s. One structural feature that likely impacts the ability of the P450s to use biosynthetic intermediates as substrates is the presence and absence of an oxygen atom attached to carbon atom 3 of EPT. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-023-12950-1.

2.17 e ± 3.02 0.03 n= 3, ANOVA.a,b,c,d,e On each column, values followed by different superscript letters are significantly different (p < 0.05).*This column includes the ratio of radial growth inhibition (RI) for each strain versus that for the wild-type strain, assigning a ratio of 100% to the values observed for the wild-type strain.Supplementary File 1.Amino acid sequences used for the phylogenetic study illustrated in Fig. 3

Fig. S1 .
Fig. S1.Examples of chemical structures of type A, B, C and D trichothecenes.The positions and/or functional groups upon which the classification system is based are highlighted with blue shading.

Fig. S2 .
Fig. S2.Schematic pathway showing the step in the biosynthesis of Fusarium trichothecenes catalyzed by TRI11 and TRI13, two cytochrome P450 monooxygenases that have been analyzed in the present work (blue shaded).Enzymes and enzymatic activities are written in uppercase black and blue letters, respecitvely.

Fig. S4 .
Fig. S4.Agarose gel electrophoresis of PCR reactions carry out to detect transformants of Dtri22 mutant that have incorporated the different constructs designed for overexpression of: A. T. arundinaceum tri22; B. P. roridum tri22; C. F. sporotrichioides tri13; D. F. graminearum tri11; and E. F. longipes tri11.F. gel electrophoresis of PCR reactions to detect transformants of F. sporotrichioides Dtri13 mutant that have incorporated P. roridum tri22 gene.Size of the expected fragments for each PCR reaction are shown at the right of each agarose gel.Transformants selected on each experiment were squared with a black lined rectangle.Oligonucleotide pairs used in the different analyses are indicated in the text.l/PstI, lambda phage DNA digested with the endonuclease PstI, used as a DNA size marker.

Fig. S7 .
Fig. S7.Differential mass spectra for m/z 124 ion, which specifically picks out sambucinol, observed from broths of DFsTRI13 (Dtri13) mutant (left panel) and transformants of that strain expressing Paramyrothecium roridum tri22 gene (Prtri22) (right panel), in both cases amended with EPT.Note that sambucinol was detected at much higher level in the mutant without P. roridum tri22.

Fig
Fig. S9. A. Chemical structure of deoxysambucinol and sambucinol.B. Effect of deoxysambucinol and sambucinol (1 mg/mL, 5 mg/mL, and 10 mg/mL) on R. solani growth.Plates labeled as D + S were amended with both compounds at the final concentration indicated at the left.

Table S2 .
Percentages of radial growth inhibition (RI) of R. solani by strains analyzed in the present work after 7 days of incubation after pathogen´s plug disposal. .